User:Matt Whelihan

Luciferases
Bioluminescence is the process by which living organisms convert chemical energy into photons of light and it is widely distributed throughout the animals, plants and fungi. Species use bioluminescence as a survival tool in mating, defense and hunting. The one thing that all bioluminescent species have in common is that they all catalyze the reaction with an enzyme generically called a luciferase. All luciferases oxidize a substrate, which then decays back to the ground state while emitting a photon of light. This process is incredibly efficient with almost one photon of light produced per oxidation. While all lucifeases oxidize their substrates, the cofactors involved and reaction pathways used, vary widely.

Applications
Luciferases have become an invaluable tool in microbiology and biochemistry as a means of reporting gene expression and in-vivo/vitro chemical conditions. Since it was first cloned in 1985 by DeLuca et al., the gene coding for luciferases has been used in reporter assays to measure gene transcription and cellular morphology. Various cell lines have also been engineered to express luciferases as a measure of the oxidative state in various organs and types of diseases. Perhaps its most useful function is in the efficient detection of intracellular ATP. Luciferases are also used to detect protein-anesthetic interactions as they are susceptible to a wide range of general anesthetics.

Firefly Luciferase
One of the most studied of all luciferases is that of the Firefly. This particular luciferase enzyme is located in the light emitting organ known as the lantern in the abdomen of the beetle. Firefly larvae glow green to ward off predators and adult fireflies use this mechanism of bioluminescence to attract mates. Luciferase binds ATP/Mg+ and D-luciferin and oxidizes it to Oxyluciferin with the products of one photon of yellow-green light, pyrophosphate, AMP and CO2. The crystal structures of two firefly luciferases, the North American Firefly (Photinus pyralis) and the Japanese Firefly (Luciola cruciata) 16541080 have been solved.



Both the Japanese and North American luciferase variants consist of a single-chain 62 kDa monooxygenases with two distinct domains that are comprised of a mix of alpha and beta secondary structures. The N-terminal domain (residues 4-436) is comprised of of an antiparalell β-barrel and two β-sheets, flanked by α-helices which forms a typical Rossmann fold The C-terminal domain (residues 440-544) is comprises of a separate α-β hinge. Firefly luciferases share significant sequence and mechanistic homology with peptide synthetases and acylCoA ligases. These enzymes belong to a superfamily of adenylate-forming enzymes that catalyze activation reactions between ATP and a carboxyl group of their substrates. This group of proteins shares an identifying motif (198SerSerGlySerThrGlyLeuProLysGly207) and has been termed the “acyl-adenylate/thioester-forming” enzyme family. Despite high sequence homology, there are only seven residues that are conserved across this superfamily (Gly200, Lys206, Glu344, Asp422, Arg437, Gly446, and Glu455). These residues are believed to be integral to the binding of ATP and the formation of an adenylate compound. These residues however are located across the N and C-terminal domains, which in the structure, are too far apart to produce catalysis. This suggested suggest that the crystallized form was in the resting state of the enzyme and it was hypothesized that the C-terminal domain would close in on the active site cleft upon substrate binding. This closing of the active site combined with various hydrophobic residues seen packed around the active site suggested that catalysis may occur in the absence of water. These seven highly conserved amino acids identify the active site which is located in the large hydrophobic cleft between the two adjacent N and C-terminal domains. Cysteine

Catalysis
Firefly luciferases catalyze the formation of the luciferin-adenylate intermediate (Blue). A proton is then abstracted in a steriospecific manner from the C-4 carbon, presumably by an enzyme base believed to be Thr343. A conformational change then occurs which allows molecular oxygen addition to the newly formed anion. As the highly reactive dioxetanone intermediate decays to the ground state, it releases a photon of light.



One of the most intriguing parts of this mechanism is about how various firefly species are able to emit different types of light with the same exact reaction. It had been hypothesized for 20 years that the enol form was responsible for the typical yellow green spectra at neutral pH by an enzyme assisted tautomerization. The keto form of the intermediate which is seen at low pH gave rise to the red spectra, presumably due to a less stable charge transfer leading to a less efficient conversion into the lower energy red Various mutational studies by Branchini et al. showed that seven residues His245, Phe247, Arg218, Ala348, Gly341, Asp422 and Thr343 were responsible for controlling the resonance-based charge delocalization of the anionic keto form of oxiluciferin. This control of resonance charge on the molecule is paramount in the enzymes ability to catalyze the yellow green emission of light following oxidation of the substrate. Various species have evolved with subtle variations in their active site structures which in turn yields a large spectral shift as the keto form of the oxyluciferin is favored.